In pure form, nitric oxide (NO) is a highly reactive gas having limited solubility in aqueous media (see U.S. Pat. No. 6,164,276). Therefore NO is difficult to introduce into biological systems without premature decomposition. Consequently, administration is typically in the form of a prodrug which is metabolically degraded to release NO. However, diseases affecting the respiratory tract may be treated by direct inhalation of gaseous NO (IgNO), diluted with an inert carrier gas. In such cases, not only must the potential hazards noted below be avoided, but in many respiratory diseases the bronchioles and alveoli may be constricted by smooth muscle contraction or obstructed by inflammation, thereby reducing access of the therapeutic gas to the desired location(s).
In vivo, NO exhibits multiple effects in a variety of tissues, its range of biological functions including smooth muscle relaxation, neurotransmission, down-regulation of NFκB production to regulate the immune response and to inhibit leukocyte adhesion, regulation of cellular oxygen consumption through effects on mitochondrial respiration, inhibition of platelet aggregation, as well as influencing host defence and cellular differentiation (see Lindwall, R B I, “Respiratory Distress Sydrome: Aspects of Inhaled Nitric Oxide, Surfactant and Nasal CPAP”; PhD thesis, (2005; pub. Karolinska Inst., Div. Anaesthesia and Intensive Care; Stockholm, Sweden)).
NO is synthesized by both constitutive and inducible isozymes of the nitric oxide synthases (NOS), which catabolize L-arginine to NO and citrulline. Endothelial constitutive NOS (eNOS), present in the walls of bronchioles and pulmonary arterioles provide NO at nanomolar concentrations for regulating vessel tone. Isozymes of inducible NOS (iNOS) are present in many cell types; upon activation they temporarily produce NO at micromolar concentrations, an activity which, under pathological conditions, has been associated with production of superoxides, peroxynitrites, inflammation and cellular damage.
Endogenously induced NO oxidizes the iron atom of a haem moiety in the enzyme soluble guanylate cyclase (SGC) in the smooth muscle cells of the lower respiratory tract airways, in the pulmonary arteries and in the membranes of circulatory platelets, thereby activating the SGC. The activated SGC forms the second messenger cGMP, which in smooth muscle cells promotes calcium-dependent relaxation, causing vasodilation of blood vessels in the lower respiratory tract, thereby increasing blood flow through the pulmonary arteries and capillaries, and also dilation of the airways in the lower respiratory tract, thereby improving bulk gas transport into the alveoli and exchange of O2 and CO2. A further result is reduction of platelet aggregation on irregular surfaces (such as a constricted blood vessel) thereby lowering the probability of thrombosis (see WO 95/10315 A1).
Other functions of NO are as a neurotransmitter in the brain where it mediates the actions of the excitatory neurotransmitter glutamate in stimulating cGMP concentrations, and in the intestine where it promotes neuronal relaxation. NO also forms nitrosyl derivatives of tyrosine residues in certain functional proteins. However, tyrosine nitration, resulting from reaction of protein tyrosine residues with NO2 or the peroxynitrite anion, is used as an indicator of cell damage, inflammation and NO production. In many disease states, oxidative stress increases the production of superoxide (•O2−).
The toxicity of IgNO is associated with a variety of properties.    (a) Firstly, NO is swiftly absorbed by lung tissue and enters the blood stream, where it reacts very rapidly with haemoglobin, oxidizing the iron atom of one of the four haem moieties to the ferric form, thereby creating stable methaemoglobin (+ nitrite and nitrate ions). Methaemoglobin's three ferrous haem groups have far greater affinity for oxygen than the haemoglobin haem moieties, so that blood in which the proportion of methaemoglobin is elevated releases insufficient oxygen to the tissues.    (b) Secondly, in the presence of oxygen NO reacts rapidly to form nitrous oxide (NO2), itself a toxic molecule. Gaseous NO2 at 5 ppm is considered to be a dangerously toxic concentration, compared to standard administrations of IgNO at 10 to 120 ppm. As lung disease frequently causes reduced respiratory function, patients are often administered an O2-enriched air supply. In the presence of such an increased concentration of O2 the probability of NO being oxidized to toxic NO2 is correspondingly greater.    (c) Thirdly, NO reacts with superoxides to form toxic peroxynitrites, powerful oxidants capable of oxidizing lipoproteins and responsible, as are both NO and NO2, for nitration of tyrosine residues. Peroxynitrite reacts nucleophilically with carbon dioxide, which is present at about 1 mM concentrations in physiological tissues, to form the nitrosoperoxycarbonate radical. This, in turn, degrades to form carbonate radical and NO2, both of which are believed to be responsible for causing peroxynitrite-related cellular damage. Nitrotyrosine is used as an indicator of NO-dependent nitrative stress induced in many disease states, generally being absent or undetected in healthy subjects.
Since, in the presence of oxygen, the NO concentration determines the production rate of NO2, over-delivery of NO will generate excessive quantities of toxic NO2. Even if inhaled for only a short period, excess NO may form sufficient methaemoglobin to reduce oxygen delivery to the tissues to dangerously low levels, particularly in patients suffering from lung disease. Excess inert carrier gas accompanying administration of IgNO may deplete oxygen content of respiratory gas supply. On the other hand, under-administration of IgNO to patients requiring relaxation of the smooth muscles in pulmonary arteries may result in excessively high arterial blood pressures causing a low partial pressure of O2 in alveolar blood (low PAO2). Consequently, precise control of the NO dosage is required at all times during administration of IgNO, in spite of irregular patient breathing patterns, fluctuations in ambient temperature and pressure, and depletion of the gas reservoir.
IgNO may be used to relax smooth muscle control of pulmonary arteriole diameter, for treating pulmonary hypertension in diseases such as acute respiratory distress syndrome (ARDS), in which impaired gas exchange and systemic release of inflammatory mediators (‘acute phase proteins’ and cytokines, particularly interleukins) cause fever and localised or systemic increases in blood pressure. IgNO will also relax smooth muscle control of bronchiole diameter, for treating emphysema in cases of ARDS and chronic obstructive pulmonary disease (COPD), in which the lower respiratory tract (particularly the lung parenchyma: alveoli and bronchioles) become inflamed. In COPD airways in the lower respiratory tract narrow and lung tissue breaks down, with associated loss of airflow and lung function which is not responsive to standard bronchodilating medication. IgNO administration may therefore assist in countering the ‘pulmonary shunt’, in which respiratory disease causes deregulation of the matching of the flow of air to the alveoli with the blood flow to the capillaries, which under normal conditions allows oxygen and carbon dioxide to diffuse evenly between blood and air (see WO 95/10315 A1).
IgNO is an effective microbicidal molecule and provides the advantage for treating infections of the respiratory tract that it acts directly in situ, whereas parenteral administration of drugs requires a high dosage to address systemic dilution and hepatic catabolism. Thus, NO has been shown to be an effective agent for killing Mycobacterium tuberculosis within cysts or tuberculi in a patient's lungs (see WO 00/30659 A1). IgNO may also be administered to treat pneumonia: pulmonary infection and inflammation (see WO 00/30659 A1). Pneumonia, which may accompany other respiratory or non-respiratory disease, is an inflammatory condition of the lung primarily affecting the alveoli resulting from infection with bacteria and/or viruses, less commonly by other organisms such as fungi or parasites. Bacteria generally enter the upper respiratory tract through aspiration of small quantities of microbial cells present in the nose or throat (particularly during sleep), or via airborne droplets. Systemic sepsis or septicaemia may also result in bacterial invasion of the lungs. Viral infection may occur through inhalation or distribution from the blood; in the lungs cells lining the airways, alveoli and parenchyma are damaged, and may render the patient more susceptible to bacterial infection of the respiratory tract. Response by the immune system to a respiratory tract infection may cause further damage through inflammation, particularly if the infection and the corresponding inflammation affect the lower respiratory tract. Macrophages and neutrophils located between pulmonary cells are mobilized to engulf and inactivate invading bacteria. The neutrophils also release cytokines, stimulating the immune response further. Fluid from surrounding blood vessels and from damaged cells, and containing defensive monocytes and invasive bacteria, flows into the alveoli in affected parts of the lung, thereby restricting influx of respiratory gas to the affected alveoli and reducing gas exchange efficiency, potentially causing a ‘pulmonary shunt’.
Cystic Fibrosis or mucoviscidosis, (‘CF’) is an autosomal recessive disorder that critically affects the lungs, but also the pancreas, liver and intestine. CF generally arises from a frameshift mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) in ciliated epithelial cells. The CFTR protein is inactivated, causing abnormal transport of sodium and chloride ions across the affected epithelia, so that secretions throughout the respiratory, alimentary and urogenital tracts become abnormally viscous. Inflammation, scarring and cyst formation occur in the pancreas due to reduced ability to mobilize the pancreatic secretions having increased viscosity. The inability of epithelial cilia to move the abnormally viscous secretions outwards for expulsion from the body causes repeated and persistent lung infections with associated chronic inflammation, pneumonia and injury to the lungs; sinus infections and infertility may also occur. Structural changes to the lungs and major airways ultimately result from the repeated infections, accompanied by hypoxia, pulmonary hypertension, respiratory failure and heart failure. The three most common bacterial lung infections in CF patients are Staphyococcus aureus, Haemohilus influenzae and Pseudomonas aeruginosa. Breathing difficulties are often aggravated by allergic bronchopulmonary aspergillosis (‘ABPA’) arising from infection with Aspergillus fumigatus, or other filamentous fungi. Mycobacterial infections of the lungs are also frequently associated with CF. Chronic administration of antibiotic and corticosteroid therapies provides conditions selecting for resistant bacterial and fungal growth, while the defective mucociliary clearance may serve to protect bacterial and fungal infection from both endogenous defence mechanisms and exogenous therapies.
Damage to the pancreas arising from CF may result in a reduction of insulin-synthesizing capacity through loss of islet cells and development of CF related diabetes (‘CFRD’). Liver disease resulting from increased viscosity of bile is a further CF complication, which may result in cirrhosis and loss of functions such as toxin catabolism and synthesis of blood clotting proteins. Children suffering from CF grow poorly due to reduced nutrient absorption through the viscous mucus of the alimentary tract and from the effects of chronic infection. In CF the alimentary tract is particularly poor at absorbing Vitamin K, causing a potential reduction in the blood clotting rate of CF patients to dangerous levels.
A number of complications are commonly associated with respiratory disease, yet have the potential to be ameliorated by administration of IgNO. In respiratory disease the bronchioles and alveoli may become obstructed or constricted by smooth muscle contraction or inflammation. Reduced access of respired gas to constricted bronchioles and alveoli reduces exchange of oxygen and CO2 between the respiratory gas and the pulmonary blood supply. In lung diseases such as asthma, pneumonia, bronchitis and emphysema, the lungs' small peripheral arteries—the pulmonary arteries—constrict under conditions in which the oxygen tension falls, causing pulmonary hypertension. In primary pulmonary hypertension the increase in resistance to the flow of blood through the pulmonary arteries and capillaries may be sufficient to cause the heart's right ventricle to fail. A further potential complication is dysregulation of the flow of air to the alveoli which is normally matched to the blood flow to the capillaries, allowing oxygen and carbon dioxide to diffuse evenly between blood and air (see WO 95/10315 A1). This uncoupling of smooth muscle regulation of the respective diameters of the airways in the lungs and of the pulmonary capillaries results in a “pulmonary shunt”: the supply of respiratory gas is no longer controlled in proportion to the blood flow through the capillaries in the affected part of the lungs. The result is normal perfusion with blood while the supply of air is restricted. Lower O2 concentrations in the blood and tissues, and especially higher CO2 concentrations, increase the pulse rate, placing more stress on the heart, which may already be compensating for pulmonary hypertension. Applying an increase in the pressure of supplied respiratory gas, as in CPAP may assist in reducing influx of fluid into the alveoli in cases of pulmonary inflammation, may assist in transfer of oxygen from respiratory gas into the blood-stream. However, where the increased pressure of the respiratory gas supply is inadequate to improve oxygen exchange either generally or in particular regions of an affected lung, any ‘compensatory’ increase in pulmonary hypertension may worsen the prognosis.
The surface barriers of the nose, mouth and the mucus secreted into the respiratory tract aid in protection of the tract against infection. However, inhibition of mucus expulsion by reduced movement of the cilia of the epithelial cells at the surface of the respiratory tract or by thickening of the mucus, as in cystic fibrosis, may result in infection occurring within the mucus itself. The innate immune response to infection includes inflammation, with longer term protection being provided against repeat infection by the cell-mediated and humoral components of the adaptive immune system. Although macrophages provide the primary cellular defence against infection macrophages themselves may undergo parasitic infection by fungal, bacterial and viral infections which may then result in disruption of the immune response to infectious agents (e.g.; due to impairment of antigen presentation) and further distribution of the infection.
Endogenously produced NO is partially responsible for the cytotoxic actions of macrophages. The mechanisms discussed above relating to potential cell damaging activities of NO supplied either endogenously or exogenously, such as the production of superoxide, the nitration of tyrosine residues in critical proteins, and the stable binding to haem groups by NO to inhibit electron transport pathways and energy metabolism, are all mechanisms which will also apply to the activity of NO in countering infection. NO being an effective microbicidal molecule, IgNO offers the advantage for treating infections of the respiratory tract that it acts directly in situ, whereas parenteral administration of drugs requires a high dosage to address systemic dilution and hepatic catabolism.
Furthermore, NO reduces the abnormally high viscosity of mucus occurring in the lower respiratory tract of CF patients, allowing ciliary activity to be restored and excess mucus to be rapidly cleared from the respiratory tract (see U.S. Pat. No. 8,518,457 B2). Thus, a combination of IgNO administration with CPAP provides an opportunity for the increased pressure of respiratory gas to counteract the reduced efficiency of gas exchange under conditions of pulmonary inflammation, where the IgNO counteracts the pulmonary shunt by relaxing smooth muscle control of bronchioles and arterioles to cause dilation of these airways and blood vessels. Thus, each of the two aspects of this treatment employing IgNO+CPAP enhances the therapeutic effect of the other, resulting in an unexpectedly effective treatment. Additionally, IgNO provides the microbicidal activity of NO for treating infections located in the lower respiratory tract, which may be a cause of or an accompaniment to the respiratory disease requiring treatment.
Miller et al., Gaseous nitric oxide bacterial activity retained during intermittent high-dose short duration exposure, Nitric Oxide 20 (2009) 16-23 describe the use of nitric oxide as an anti-infective agent for non-healing wounds due to its non-specific antimicrobial properties.
Using gaseous nitric oxide as an inhalable medicament for the treatment of reversible pulmonary vasoconstriction or bronchus constriction is known. Nitric oxide is usually administered to mammals suspected of having acute pulmonary vasoconstriction at a concentration of from 1 ppm to 40 ppm in air, pure oxygen or another suitable gas or gas mixture for as long as needed. This is disclosed, for example, in EP 0 560 928 B1.
Nitric oxide is also known as a mucolytic agent wherein the nitric oxide is provided in concentrations of about 160 ppm to about 220 ppm to the breathing gas, for example air. This is disclosed, for example, in U.S. Pat. No. 8,518,457 B2.
A controlled gas supply system for supplying nitric oxide to the breathing air to a patient is known from EP 0 973 443 B1.
Furthermore, in order to recruit additional lung units for the gas exchange it is known to use a positive airway pressure (CPAP) device, mainly in order to treat obstructive sleep apnea.
Christian Putensen et al: Positive airway pressure modulates effect of a nitric oxide on the ventilation perfusion distributions in canine lung injury, Chest, 106, 5, Nov. 1994, page 1563 to 1569, suggest using a combination of the application of positive airway pressure and the inhalation of nitric oxide in concentrations ranging from 5 to 80 ppm to cause selective pulmonary vasodilation and to improve pulmonary gas exchange in patients with acute lung injury.